Hearing and balance disorders are some of the most common disabilities in the United States, with 0.1% of newborns having some form of hereditary hearing loss and roughly half of all adults suffering from some degree of hearing loss by the time they reach retirement age. The causes of age-dependent hearing loss are complex, with both genetic and environmental factors playing a role. The most common reason for hearing loss is the death of sensory hair cells in the inner ear. In addition to genetic factors, damage from noise exposure or from certain kinds of antibiotics and chemotherapy drugs can kill hair cells. Once hair cells die in mammals, they are never replaced and hearing loss is therefore permanent. Many experiments over the last 15 years have suggested that the transcription factor Atoh1 plays a central role in the development of sensory hair cells. First, it is one of the earliest genes to be expressed in differentiating hair cells. Second, inactivation of Atoh1 in mice causes a complete failure of hair cell differentiation in both the cochlear and vestibular sensory organs. Third, Atoh1 is one of the first genes to be expressed during hair cell regeneration in non-mammalian vertebrates. Finally, ectopic expression of Atoh1 is sufficient to generate new hair cells in some regions of the inner ear in vitro and in vivo. For these reasons, Atoh1 has been proposed as tool for gene therapy to replace lost auditory or vestibular hair cells. Despite the great interest in Atoh1's function in development and its potential use in regenerative therapies, very little is known about the genes that are regulated by Atoh1 in hair cells. At one extreme, Atoh1 might regulate a relatively small number of other transcription factors, which would each regulate different aspects of hair cell development, maturation and survival. Alternatively, Atoh1 might have a much broader role in directly regulating many aspects of hair cell structure and function. Moreover, it is not clear whether Atoh1 transcriptional targets differ between auditory and vestibular hair cells, or whether these differences are determined by other factors, with Atoh1 simply regulating generic hair cell properties. In this pilot project, we propoe to use cutting-edge deep sequencing and bioinformatic approaches to identify the direct targets of Atoh1 in hair cells - the so-called Atoh1 targetome. We will work in collaboration with Dr. Huda Zoghbi at Baylor College of Medicine, whose lab recently used similar technology to successfully characterize the Atoh1 targetome in cerebellar granule cells. A gene that is directly regulated by Atoh1 should possess three attributes - it should be expressed in an Atoh1-dependent manner, the genomic locus of the gene should be transcriptionally active, and Atoh1 protein should bind to regulatory regions adjacent to the gene. The successful strategy of the Zoghbi lab involved a genome-wide analysis of these three properties, coupled with statistical ranking to generate a list of direct Atoh1 targets. We now propose to apply these same methods to identify the Atoh1 targetome in hair cells. PUBLIC HEALTH RELEVANCE: Sensory hair cells are specialized cells in the inner ear that detect sound and mediate our sense of balance. This project will identify genes that are important for the development and survival of sensory hair cells. The long term goal is to use this information to design therapies for individuals who suffer hearing or balance disorders due to the loss of sensory hair cells.